Interaction between different leukocyte subsets and stromal cells in different organ microenvironments is critical for optimizing organ-specific immune responses that could limit metastasis formation and control disease-induced inflammation. IL-12 and IL-18 are potent immunoregulatory cytokines for natural killer (NK), NKT, and T cells, and have very distinctive effects on the NKT/NK subsets in different organs. The liver is a major target for the formation of metastases. We used a mouse model of liver metastasis to study the anti-tumor activity of IL-18 and/or IL-12 cytokines and the roles of NK and NKT cells to these responses. Treating mice with IL-18 plus IL-12 significantly reduced the number of tumor nodules in the liver to a greater degree than did either of the cytokines alone. As expected, IL-18 plus IL-12 stimulated a synergistic increase in systemic IFN-gamma in tumor bearing mice. The anti-tumor activity of IL-18 plus IL-12 therapy was abolished in IFN-gamma(-/-) mice. Using intracellular staining, NK and NKT cells were identified as the major producers of IFN-gamma in the livers of mice treated with IL-18 and/or IL-12. Liver NK cells were increased with daily treatment of mice with IL-18 plus IL-12 whereas liver NKT cells were diminished by this treatment. Preferential depletion of NK cells with anti-asGM1 resulted in a partial loss of the anti-tumor activity of IL-18 plus IL-12 therapy and revealed NK cells to be an important component of the mechanism for tumor regression. In contrast, the preferential depletion of NKT cells with betaGalCer decreased the number of liver tumor nodules in mice treated with vehicle control or IL-18 alone. Similarly, all treatment approaches showed increased anti-tumor activity in CD1d(-/-) mice, which lack NKT cells. Our data therefore shows that the IL-18 plus IL-12 induced anti-tumor activity in mice is NK and IFN-gamma dependent, and is able to overcome an endogenous immunosuppressive effect of NKT cells. These results thereby suggest that immunotherapeutic approaches that enhance NK cell numbers and function while preferentially depleting NKT cells could be effective in the treatment of cancer in the liver. Because IL-18 plus IL-12 treatment of tumor bearing mice elicited significant anti-tumor activity concurrent with a decrease in the detectable number of invariant NKT (iNKT) cells, we compared iNKT cell modulation mechanisms of alpha-GalCer, a glycolipid known to modulate iNKT cells, and IL-18 plus IL-12. We found acute treatment with IL-18 + IL-12 induced a loss of NK1.1(+) subset of iNKT cells, while acute treatment with aGalCer depleted all liver iNKT cells at 24hrs. However, other lymphoid tissues showed little to know loss of these cells suggesting the liver microenviroment is a critical regulator of iNKT cells. At 72hrs iNKT cells repopulated and expanded in the liver following aGalCer and IL-18 plus IL-12 treatment of mice. To study this expansion, we administered BrdU to mice 24hrs after acute treatment with IL-18 plus IL-12 or aGalCer and found a high percentage of proliferating iNKT cells. We further found the spleen was needed for iNKT repopulation and expansion following aGalCer, but not for IL-18 plus IL-12 treatment, as no expansion was observed in splenectomized mice. We next examined chronic treatment of mice with aGalCer or IL-18 plus IL-12 and found aGalCer induced a severe loss of liver iNKT cells, while only IL-18 plus IL-12 caused a systemic loss of iNKT cells. Long term (&gt;60 days) thymic development was needed to repopulate the liver following both treatments. These data reveal an important role for the liver microenvironment in regulating iNKT cell response to inflammatory signals.